Poor ischemic tolerance and loss of ischemic preconditioning are hallmarks of the aged heart, and contribute to the fact that age is the strongest risk factor for cardiovascular disease. However, the metabolic adjustments to a diet that is calorie restricted (CR) prevents these deleterious effects of aging. Our overall hypothesis is that CR changes the abundance and proliferative state of: 1) resident progenitor cells in the heart, and 2) adipose- derived stem cells that are used to treat peripheral vascular disease, ischemic heart disease and intractable angina. This hypothesis is supported by data demonstrating that CR not only retards aging, but also increases the abundance and proliferative capacity of resident stem cells in some tissues. The long term goal of this research is to exploit the established anti-aging effects of CR to improve cardiac health in patients. These studies will also provide insight into the broader questions of whether diet might influence health via effects on resident stem cells. The first aim is to test whether CR increases the proliferative state of a specific type of cardiac progenitor cell, cardiac side population cells that are Sca1? . We focus on this specific cell type because it is a distinct population that consists of progenitors for a cell type (cardiomyocytes) that likely play a central role in the poor ischemic tolerance and other deleterious aspects of cardiac aging. The second aim is to define the impact of CR on the abundance, proliferative state, and colony forming of adipose-derived stem cells. We have chosen to focus on adipose tissue (epididymal fat pad) as a source of donor cells because it undergoes extensive remodeling during CR, and because of the literature demonstrating the efficacy of these cells to revascularize ischemic tissue and treat myocardial infarctions. Male C57Bl/6 mice will receive a control diet or a CR diet (60% of ad libitum calories) from 6 months of age until they are sacrificed at middle age (12 months) or senescence (24 months of age). This will allow us to determine the impact singly, and in combination, of aging and CR. Fluorescence activated cell sorting will be used to identify cardiac side population cells that are lineage negative/Sca1? (cardiomyocyte progenitor cells), and adipose-derived cells that are lineage negative/CD34? (stem cells with cardiomyogenic potential). The effects of age and CR on the proliferative state of these cells will be assessed by measuring their rates of BrdU incorporation, telomerase activity, and the abundance of molecular markers of cellular division (Ki67) and cell cycle arrest (p16INK4a). To assess the overall proliferative state of cardiomyocyte precursors the rate of new cardiomyocyte formation (BrdU incorporation into cardiomyocytes) and cardiomyocyte age (telomerase activity) will be measured. If CR increases the number and proliferative state of these two types of resident stem/progenitor cells, the proposed studies will form the basis for future studies determining how best to exploit these effects of CR for clinical use. PUBLIC HEALTH RELEVANCE Aging can be slowed and lifespan extended by placing animals on a diet that is very low in calories and enriched in vitamins and minerals. How this type of diet slows aging and prevents age-associated diseases is not known. Our goal is to determine if this diet "rejuvenates" stem cells located in the adult heart and fat.